Project Summary/Abstract Cataracts, defined as any opacity in the eye lens, remain the leading cause of blindness in the world despite the success of cataract surgeries. Decades of studies have increased our knowledge about cataract formation, but the underlying mechanisms that regulate lens growth and facilitate lifelong homeostasis remain unclear. The avascular lens maintains fluid and ion homeostasis through a vast network of gap junctions that couple neighboring fibers, and decreased gap junction communication leads to congenital and age-related cataracts in humans. Recent studies have reported that mutations of the EphA2 or ephrin-A5 gene are associated with variable congenital and age-related cataracts in humans and mice, and these bidirectional signaling molecules are key components for regulating lens fiber cell organization and stability. While EphA2 and ephrin-A5 are a receptor-ligand pair in other cell types, our previous studies and our new preliminary data from EphA2 and ephrin-A5 double knockout lenses indicate that, in mouse lenses, EphA2- ephrin-A5 is not a receptor-ligand pair. Therefore, we hypothesize that ephrin-A5 interacts with another Eph receptor to maintain the anterior epithelium while EphA2 partners with another ephrin ligand to regulate the organization of equatorial epithelial cells and differentiating fiber cells. Members of Eph/ephrin protein family likely play diverse and/or redundant functions to spatially and temporally coordinate the signal transduction network to control distinct properties of lens epithelial cells and fibers. We will evaluate the spatiotemporal expression and localization of Ephs and ephrins in the lens and determine the canonical ligand-mediated bidirectional signaling and non-canonical ligand-independent Eph receptor signaling pathways are that are important for maintaining lens homeostasis. This work will identify the lens Eph-ephrin pairs and determine the downstream pathways activated by Eph-ephrin signaling in the lens. Intriguingly, our preliminary data also indicates that gap junctions may be compromised in EphA2 knockout lenses that often have mild nuclear cataracts. Our results show that gap junction plaque formation is altered in peripheral knockout lens fibers. We will study age-related changes in EphA2 knockout and ephrin-A5 knockout lenses and test whether disruptions in Eph-ephrin signaling lead to changes in gap junction communication or non-communication-related functions of connexins, including regulation of cell cycle and cell-cell adhesion, in the lens. We will determine whether knockout lenses have altered growth and fiber cell differentiation or adhesion, and/or changes connexin transcript and protein levels, connexin protein cleavage and gap junction plaque assembly/stability. Lens physiology experiments will reveal whether knockout lenses have compromised gap junction conductance and/or ion/fluid homeostasis. This data will provide a better understanding of coordinated signaling mechanisms for maintaining homeostasis in normal lenses, which may lead to the development of new non-surgical approaches to delay or prevent age-related cataract formation.